Professional Documents
Culture Documents
Les milieux de culture et leur utilisation en
contrôle qualité
Stéphanie Goineau, B.Sc Microbiologiste
Directrice des Services de Microbiologie
Siebel Institute of Technology (Lallemand Brewing)
Congrès AMBQ – Journée technique MBAA
Montréal, 21 novembre 2012
Wort VS Beer :
Culture media for contaminants ?
WORT BEER
A beer contaminant must
be resistant to this harsh
environment !
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Contaminants
What are they?
y Where do theyy come from?
y Bacteria y Raw materials
y Yeast y Water
y Fungi y Environment
y Airborne
y Insects
y Repitched yeast
y Etc.
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Contaminants detection :
Why is it so important to have QC procedures?
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Detection & Identification
1. Aseptic sampling & techniques
2. Choose the right media (duplicate or triplicate)
3. Choose the appropriate plating technique
A. Spread plate method
B. Pour plate method
y Both methods recommended for samples in which high levels of contamination is
expected : yeast, beer and ingredients such as process water
y Dilutions might be required ( 30‐300 colonies /plate = significantly representative)
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Plating techniques
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Different types of media
y Nutritive medium: Synthetic, complex medium prepared in the lab
h i i f diff h i l h
that contains various amounts of different chemicals that are
known to support growth of many microorganisms.
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Different types of media…
y Inhibitory medium (I):
One which inhibits certain
microorganisms from growing
while allowing others to grow –
usually by adding an inhibitory
substance (like antibiotics).
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General guidelines on
how to prepare media?
y Follow manufacturer’s instructions!
y Heat
H t to boiling
t b ili until
til complete
l t dissolution
di l ti
y Let media cool down to approx. 50C
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General guidelines on
how to prepare media…
y Add inhibitory agents if necessary – sterile stock solutions!
y Let it solidified
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Bacteria
y Unicellular organisms
y Gram (+) or (‐)
y Various morphology
y Coccus
y Rod
y Multiple arrangements
y Single cells
y Pairs
y Tetrads
y Chains
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Enterobacteriaceae
« coliforms »
y Gram (‐), short straight rods
y Facultative anaerobe
y RISK: Wort, early fermentation
y SPOILAGE:
y Diacetyl
y Organic acids
y Phenolics
y CONTROL:
y Sensitive to pH < 4.4 (beer pH 4.5)
y Sensitive to ETOH > 2%
y Heat sensitive (pasteurization)
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Anaerobic bacteria ‐
Zymomonas
y Gram (‐), short straight rods, rosettes
( ), g ,
y Anaerobic, but oxygen tolerant
y Ferment glucose & fructose, not maltose
y SPOILAGE:
y H2S
y Acetaldehyde (green apple)
y Alcohol
l h l tolerant
l at very high
h h levels
l l (ETOH > 13%)
( )
y CONTROL:
y Hygiene
y CIP
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General Culture Media
for the brewery
UBA Included in the ASBC « Methods of
Analysis »
(Universal Beer Agar)
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General Culture Media
for the brewery
LMDA / SDA
(Lee’s Multi‐Differential Agar
Included in the ASBC « Methods of Analysis »
/ Schwartz Differential Agar)
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General Culture Media
LMDA / SDA …
y A bi and/or
Aerobic d/ anaerobic
bi incubation
i b ti
at 28oC :
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General Culture Media
for the brewery
WLN WLD
(Wallerstein Nutrient Agar)
y WLN is a good media to grow y WLN that has been made
brewer’s yeast. selective for bacterial
contaminants by the addition
y Differential medium that can be of actidione.
used to evaluate macroscopic
characteristics / differences of
yeast colonies (variants). y Incubate aerobically 2‐3 days
for acetic acid bacteria
y For yeast growth, incubate at 28‐
30oC for 24‐48 hours and up to 4
weeks for the giant colonies assay. y Incubate anaerobically for 4‐6
days for LAB investigation
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General Culture Media
WLN
Lager yeast strain Ale yeast strain
(Powell & Diacetis, 2007) (P ll & Di ti 2007)
(Powell & Diacetis, 2007)
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How to detect LAB ?
HLP
(Hsu’s Lactobacillus/Pediococcus)
Included in the ASBC « Methods of
S i lid medium
Semi‐solid di h
that bl
enables Analysis »
selective detection & quantification of
lactic acid bacteria. Free video on Siebel’s website &
YouTube
Many LAB can be detected in 2 days
(28oC). Differentiation of Lactobacillus HLP PLATES
and Pediococcus can be made after 5‐ ‐ Add 1.5‐2.0g agar
7 days.
‐ Anaerobic incubation needed !
Yeast growth is suppressed by
cycloheximide already in the medium.
How to detect LAB?
HLP (Hsu’s Lactobacillsus / Pediococcus)
Lactobacillus sp.
p Pediococcus sp.
p
y Inverted tear drop shape colonies y Comet shape colonies
y Rain drop shape colonies
y Snowballs shape colonies
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How to detect LAB ?
RRLM
(Raka‐Ray Lactic Acid Bacteria Medium)
y Selectively detects LAB in beer and brewing process by encouraging larger colonies of
this bacterial group to grow in a shorter time.
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How to detect LAB ?
BMB
(Barney‐Miller Brewery Medium)
y Time required for incubation has been reduced compared to other selective media
for LAB detection
y Growth in +/‐ 3 days for most LAB but up to 7 days for slow‐growing lactics
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How to detect LAB ?
MRS
(
(De Man Rogosa h )
Sharpe)
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How to detect enterics ?
MacConkey Agar
y Selective medium for Gram (‐) enterics because crystal violet and bile salts
inhibit Gram (+) bacteria.
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How to detect
Megaspherea and Pectinatus ?
SMMP (Selective Medium Megaspherea and Pectinatus)
Megaspherea and Pectinatus
SMMP…
Megaspherea Pectinatus
y Coccoid cells, large y Rods
y Medium may change from y Medium remain purple with
purple to yellow after a longer sediments
incubation period y Produces acetic and propionic
y Produces butyric, isovaleric, acids
valeric, caproic and caprylic
acids
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WILD YEASTS
Any yeast that has not been
d lib l introduced
deliberately i d d in i
the wort, including other
production strains…
1) Non‐Saccharomyces
2) Saccharomyces
Important to use a
combination of media for
optimal detection of wild
yeasts.
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Non‐Saccharomyces Wild Yeasts
y Ellipsoidal to brick
y Aerobes
y Various genus: Pichia sp., Hansenula sp.,
Candida sp., Brettanomyces sp.,
Schizosaccharomyces sp.
y RISK: Fermentation onwards
y SPOILAGE:
Form films in the presence
y F fil i h of air
f i
y Haze
y Off flavors
y Pichia & Candida: reduction in ETOH concentration : ETOH ‐> acetic acid
y Brettanomyces: acetic acid, mousy flavor
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How to detect
non‐Saccharomyces Wild Yeasts?
LCSM Selective medium for the
(Lin’s Cupric Sulfate Medium) detection and quantitative
determination of wild yeast
populations in brewer’s culture
yeast.
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How to detect
non‐Sacch Wild Yeasts ? LCSM…
y Important to use the CS
solution from the same lot
than the dehydrated LCSM
powder
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How to detect
non‐Saccharomyces Wild Yeasts?
LYSINE MEDIUM Selective medium in which lysine is
nitrogen While
the sole source of nitrogen.
most Saccharomyces sp. are lysine‐
negative, many other yeasts can
utilize it and will therefore grow on
this medium.
y at
Aerobic incubation 2‐6 days
25‐28C
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How to detect
non‐Saccharomyces Wild Yeasts?
CLEN medium
y ASBC recommended
y Multinitrogen source medium that utilizes cadaverine, lysine, ethylamine, and nitrate as
nitrogen sources.
y It has been observed that some species of WY developed as larger colonies on CLEN than on
the other WY media
y It has also been observed that CLEN medium exhibited the best recovery (highest count) for
some WY species than the other WY media
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Saccharomyces Wild Yeasts
y Ellipsoidal, discrete or chain forming
y Facultative anaerobes
y RISK: fermentation onwards
SPOILAGE – S. ellipsoidus:
y Slow sedimentation
y No interaction with finings (no clumping)
p
y Resist pasteurization
SPOILAGE – S. diastaticus:
y Superattenuation: breaks down maltotriose and dextrins to
produce ETOH and CO2
y POF
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How to detect
Saccharomyces Wild Yeasts?
LWYM Selective medium for the
detection and quantitative
(Lin’s Wild Yeast Medium) determination of wild yeast
populations in brewing culture
yeast.
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How to detect
Sacch Wild Yeasts? LWYM…
y Wild
d yeast will show
s o vigorous
go ous ggrowth
o t and
a d
grow as larger distinct colonies
Saccharomyces Wild Yeasts…
y SPOILAGE – Petite / RD mutants :
y Result from genetic drift within culture yeast population
y Lack certain respiratory enzymes
y Associated with poor growth, lower yeast viability and unfavorable
flavor production
y Poor flocculation
y Slow fermentation
y SPOILAGE
SPOILAGE – Killer yeasts:
Killer yeasts:
y Toxin ‐> disrupts plasma membrane of sensitive yeasts = DEATH !
y Poor flocculation
y POF
y Superattenuation
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How to detect
Saccharomyces Wild Yeasts?
Respiratory Deficient Mutants : TTC Overlay technique
Add TTC
YM or YPD agar +CHL
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Identification of contaminants… briefly
BACTERIA YEAST
y Gram stain : (+) or (‐) y Microscopic evaluation
y Microscopic evaluation:
y Cell shape
y Cell shape, arrangement
y Gram y Budding
y Catalase test y C sources assimilation /
y Oxydase test fermentation test
y C sources assimilation/
y Genus & Species
& Species ID
fermentation tests
y Genus & Species ID y Api galleries (20C AUX, ID32)
y Various api galleries y Biolog
y Biolog y Genetic ID methods
y Genetic ID methods
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CONCLUSION & Suggestions
y Which media should you choose?
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Useful References
y ASBC: http://www.asbcnet.org/
p // g/
y Difco / Becton Dickinson (BD) : http://www.bd.com/
y EMD: http://www.emdchemicals.com/
y Fisher Scientific:
http://www.thermofisher.com/global/en/home.asp
y HiMedia: http://www.himedialabs.com/abouthimedia.aspx
y Neogen / Acumedia: http://www.neogen.com/acumedia/
/ d h // / d /
y OXOID: http://www.oxoid.com/uk/blue/index.asp
y Siebel Institute : http://www.siebelinstitute.com/
y VWR Canlab: https://www.vwrsp.com/
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